Free energy of hydration of collagen models and the enthalpy of the transition between the triple-helical coiled-coil and single-stranded conformations

Interactions with water make an important contribution to the free energy of stabilization of the collagen triple helix, but they do not alter the structure of the triple helix, i.e., the packing geometry of the three strands. Conformational energy computations have been carried out on poly(tripeptide) analogues of collagen, with the introduction of a newly developed form of a hydration shell model to compute the free energy of hydration. The most stable triple helix formed by poly(Gly-Pro-Pro), obtained earlier from conformational energy computations [M. H. Miller & H. A. Scheraga (1976) J. Polym. Sci. Polym. Symp. 54, 171], with a structure that is very closely similar to the observed structure, is strongly favored over other three-strand complexes, both in the absence and the presence of hydration. The hydration shell model does not provide an explanation for the increased stability of the poly(Gly-Pro-Hyp) triple helix as compared to poly(Gly-Pro-Pro). It appears that the difference should be attributed to specific binding of water, and effect that is not yet included in the present version of the hydration shell model. On the other hand, this model accounts for the observed enthalpy of unfolding of a poly(Gly-Pro-Pro) triple helix to isolated single chains in solution in terms of intramolecular noncovalent interactions and the free energy of hydration.     

DNA as a matrix of collagen fibrils

In this paper we demonstrate that DNA binds to collagen directly to form DNA–collagen complex. Our model suggests that DNA, containing well-arranged phosphate groups, helps the collagen to make ordered aggregates—fibrils. During this process hydration shell of collagen triple helix destroys and stabilizes hydration shell of ds-DNA.     

A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

Structural heterogeneity of type I collagen triple helix and its role in osteogenesis imperfecta

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (DeltaTm) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of DeltaTm with the identity of the substituting residue. Instead, we observed regular variations in DeltaTm with the substitution location in different triple helix regions. To relate the DeltaTm map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (DeltaG) from the reported peptide data. We constructed the DeltaG map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the DeltaTm and DeltaG maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the DeltaTm map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the alpha1(I) chain.     

NMR shows hydrophobic interactions replace glycine packing in the triple helix at a natural break in the (Gly-X-Y)n repeat

Little is known about the structural consequences of the more than 20 breaks in the (Gly-X-Y)(n) repeating sequence found in the long triple helix domain of basement membrane type IV collagen. NMR triple resonance studies of doubly labeled residues within a set of collagen model peptides provide distance and dihedral angle restraints that allow determination of model structures of both a standard triple helix and of a triple helix with a break in solution. Although the standard triple helix cannot continue when Gly is not every third residue, the NMR data support rod-like molecules that have standard triple-helical structures on both sides of a well defined and highly localized perturbation. The GAAVM break region may be described as a "pseudo triple helix," because it preserves the standard one-residue stagger of the triple helix but introduces hydrophobic interactions at the position normally occupied by the much smaller and hydrogen-bonded Gly residue of the repeating (Gly-X-Y)(n) sequence. This structure provides a rationale for the consensus presence of hydrophobic residues in breaks of similar length and defines a novel variant of a triple helix that could be involved in recognition.     

Carbamylation differentially alters type I collagen sensitivity to various collagenases.

Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-product of urea, on free amino groups of proteins. Post-translational modifications are known to induce alterations in structural and functional properties of proteins, thus disturbing protein-protein or cell-protein interactions. We report the impact of carbamylation on type I collagen sensitivity to enzymatic proteolysis. Type I collagen was extracted from rat tail tendons and carbamylated by incubation with 0.1 M potassium cyanate at 37 degrees C for 2, 6 or 24 h. Degradation assays revealed that carbamylated collagen exhibited a greater resistance to collagenases (i.e. bacterial collagenase, matrix metalloproteinase(MMP)-1, MMP-8 and MMP-13), together with an increased sensitivity to MMP-2. Evaluation of collagen triple helix conformation by polarimetry indicated that local destabilizations of triple helix structure related to carbamylation could be responsible for the observed differences in sensitivity. These results confirm the crucial role of triple helix integrity in the degradation of type I collagen by MMPs, and support the deleterious impact of post-translational modifications in vivo by altering the balanced remodeling of collagen within connective tissue.     

Region-specific role of water in collagen unwinding and assembly

Conformational stability of the collagen triple helix affects its turnover and determines tissue homeostasis. Although it is known that the presence of imino acids (prolines or hydroxyprolines) confer stability to the molecule, little is known regarding the stability of the imino-poor region lacking imino acids, which plays a key role in collagen cleavage. In particular, there have been continuing debates about the role of water in collagen stability. We addressed these issues using molecular dynamics simulations on 30-residue long collagen triple helices, including a structure that has a biologically relevant 9-residue imino-poor region from type III collagen (PDB ID: 1BKV). A torsional map approach was used to characterize the conformational motion of the molecule that differ between imino-rich and imino-poor regions. At temperatures 300 K and above, unwinding initiates at a common cleavage site, the glycine-isoleucine bond in the imino-poor region. This provides a linkage between previous observations that unwinding of the imino-poor region is a requirement for collagenase cleavage, and that isolated collagen molecules are unstable at body temperature. We found that unwinding of the imino-poor region is controlled by dynamic water bridges between backbone atoms with average lifetimes on the order of a few picoseconds, as the degree of unwinding strongly correlated with the loss of water bridges, and unwinding could be either prevented or enhanced, respectively by enforcing or forbidding water bridge formation. While individual water bridges were short-lived in the imino-poor region, the hydration shell surrounding the entire molecule was stable even at 330 K. The diameter of the hydrated collagen including the first hydration shell was about 14 A, in good agreement with the experimentally measured inter-collagen distances. These results elucidate the general role of water in collagen turnover: water not only affects collagen cleavage by controlling its torsional motion, but it also forms a larger-scale lubrication layer mediating collagen self-assembly.     

Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

Structural hierarchy controls deformation behavior of collagen

The structure of collagen, the most abundant protein in mammals, consists of a triple helix composed of three helical polypeptide chains. The deformation behavior of collagen is governed by molecular mechanisms that involve the interaction between different helical hierarchies found in collagen. Here, we report results of Steered Molecular Dynamics study of the full-length collagen molecule (～290 nm). The collagen molecule is extended at various pulling rates ranging from 0.00003/ps to 0.012/ps. These simulations reveal a new level of hierarchy exhibited by collagen: helicity of the triple chain. This level of hierarchy is apparent at the 290 nm length and cannot be observed in the 7-9 nm models often described to evaluate collagen mechanics. The deformation mechanisms in collagen are governed by all three levels of hierarchy, helicity of single chain (level-1), helical triple helix (level-2), and hereby described helicity of the triple chain (level-3). The mechanics resulting from the three levels is described by an interlocking gear analogy. In addition, remarkably, the full-length collagen does not show much unwinding of triple helix unlike that exhibited by short collagen models. Further, the full-length collagen does not show significant unwinding of the triple helix, unlike that exhibited by short collagen. Also reported is that the interchain hydrogen bond energy in the full-length collagen is significantly smaller than the overall interchain nonbonded interaction energies, suggesting that the nonbonded interactions have far more important role than hydrogen bonds in the mechanics of collagen. However, hydrogen bonding is essential for the triple helical conformation of the collagen. Hence, although mechanics of collagen is controlled by nonbonded interchain interaction energies, the confirmation of collagen is attributed to the interchain hydrogen bonding.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Fiber molecular model of atelocollagen-small interfering RNA (siRNA) complex

Previously we represented molecular model of collagen triple helix–DNA double helix complex [G.M. Mrevlishvili, D.V. Svintradze, Int. J. Macromol. 36 (2005) 324–326; G.M. Mrevlishvili, D.V. Svintradze, Int. J. Macromol. 35 (2005) 243–245]. We also proved that during the complex formation hydration of triple helix destroys and forms new water bridges maintaining the complex nano-structure. In this paper we demonstrate that small interfering RNA binds to atelocollagen directly to form siRNA–atelocollagen fiber complex.     

Self-association of collagen triple helic peptides into higher order structures

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.     

The Self-assembly of a Mini-fibril with Axial Periodicity from a Designed Collagen-mimetic Triple Helix

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.     

Contribution of dipole-dipole interactions to the stability of the collagen triple helix

Unveiling sequence-stability and structure-stability relationships is a major goal of protein chemistry and structural biology. Despite the enormous efforts devoted, answers to these issues remain elusive. In principle, collagen represents an ideal system for such investigations due to its simplified sequence and regular structure. However, the definition of the molecular basis of collagen triple helix stability has hitherto proved to be a difficult task. Particularly puzzling is the decoding of the mechanism of triple helix stabilization/destabilization induced by imino acids. Although the propensity-based model, which correlates the propensities of the individual imino acids with the structural requirements of the triple helix, is able to explicate most of the experimental data, it is unable to predict the rather high stability of peptides embedding Gly-Hyp-Hyp triplets. Starting from the available X-ray structures of this polypeptide, we carried out an extensive quantum chemistry analysis of the mutual interactions established by hydroxyproline residues located at the X and Y positions of the Gly-X-Y motif. Our data clearly indicate that the opposing rings of these residues establish significant van der Waals and dipole-dipole interactions that play an important role in triple helix stabilization. These findings suggest that triple helix stabilization can be achieved by distinct structural mechanisms. The interplay of these subtle but recurrent effects dictates the overall stability of this widespread structural motif.     

Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.     

Backbone Dynamics of Triple-helical Collagen-like Structure

Some details of the backbone dynamics in the collagen-like triple helix is discussed and the role of backbone dynamics in functioning collagen proteins is illustrated. On a series of oligotripeptides synthetic analogs of collagen formation of high-frequency vibrational backbone dynamics and low-frequency nonlinear backbone dynamics upon stepwise elongation of peptide chain have been described using infrared spectroscopy and hydrogen-exchange method. In the fully completed triple helix the level of high-frequency backbone dynamics is regulated firstly by contact interactions of adjacent atoms and chemical bounded groups, while the level of low-frequency large-amplitude backbone dynamics depends mainly on cooperative interactions attributed by conjugation of interpeptide hydrogen bonds. In native collagens the nonlinear large-amplitude dynamics following by non-denaturational micro-unfolding of the triple-helical structure appears to be under the natural selection control delivering an optimal condition for formation, functioning and utilization of collagen fibrils.     

Structural insights into charge pair interactions in triple helical collagen-like proteins

The collagen triple helix is the most abundant protein fold in humans. Despite its deceptively simple structure, very little is understood about its folding and fibrillization energy landscape. In this work, using a combination of x-ray crystallography and nuclear magnetic resonance spectroscopy, we carry out a detailed study of stabilizing pair-wise interactions between the positively charged lysine and the negatively charged amino acids aspartate and glutamate. We find important differences in the side chain conformation of amino acids in the crystalline and solution state. Structures from x-ray crystallography may have similarities to the densely packed triple helices of collagen fibers whereas solution NMR structures reveal the simpler interactions of isolated triple helices. In solution, two distinct types of contacts are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. However, in the crystalline state, there is a significant rearrangement of the side chain conformation allowing for packing interactions between adjacent helices, which suggests that charged amino acids may play a dual role in collagen stabilization and folding, first at the level of triple helical assembly and second during fibril formation.     

Sequence recombination improves target specificity in a redesigned collagen peptide abc‐type heterotrimer

Stability of the collagen triple helix is largely governed by its imino acid content, namely the occurrence of proline and 4R‐hydroxyproline at the X and Y positions, respectively, of the periodic (Gly‐X‐Y)_n sequence. Although other amino acids at these positions reduce stability of the triple helix, this can be partially compensated by introducing intermolecular side‐chain salt bridges. This approach was previously used to design an abc‐type heterotrimer composed of one basic, one acidic, and one neutral imino acid rich chain (Gauba and Hartgerink, J Am Chem Soc 2007;129:15034–15041). In this study, an abc‐type heterotrimer was designed to be the most stable species using a sequence recombination strategy that preserved both the amino acid composition and the network of interchain salt bridges of the original design. The target heterotrimer had the highest T_m of 50°C, 7°C greater than the next most stable species. Stability of the heterotrimer decreased with increasing ionic strength, consistent with the role of intermolecular salt bridges in promoting stability. Quantitative meta‐analysis of these results and published stability measurements on closely related peptides was used to discriminate the contributions of backbone propensity and side‐chain electrostatics to collagen stability. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.